Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.     

A finger protein structurally similar to TFIIIA that binds exclusively to 5S RNA in Xenopus

A 5S RNA binding protein (p43) in Xenopus is a major constituent of oocytes and comprises part of a 42S ribonucleoprotein storage particle. We have cloned and sequenced p43 cDNA from X. laevis and X. borealis as well as the cDNA for X. borealis TFIIIA. Like TFIIIA, p43 has nine zinc fingers, seven of which are exactly the same size as their counterparts in TFIIIA. Amino acid homology between the two proteins is restricted mainly to conserved residues characteristic of zinc fingers. In contrast to TFIIIA, which binds specifically to both 5S RNA and 5S RNA genes, p43 binds exclusively to 5S RNA.     

RNA-protein interactions of stored 5S RNA with TFIIIA and ribosomal protein L5 during Xenopus oogenesis

We studied the pathway of 5S RNA during oogenesis in Xenopus laevis from its storage in the cytoplasm to accumulation in the nucleus, the sequence requirements for the 5S RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes. In situ hybridization to sections of oocytes indicates that 5S RNA first becomes associated with the amplified nucleoli during vitellogenesis when the nucleoli are activity synthesizing ribosomal RNA and assembling ribosomes. When labeled 5S RNA is microinjected into the cytoplasm of stage V oocytes, it migrates into the nucleus, whether microinjected naked or complexed with the protein TFIIIA as a 7S RNP storage particle. During vitellogenesis, a nonribosome bound pool of 5S RNA complexed with ribosomal protein L5 (5S RNPs) is formed, which is present throughout the remainder of oogenesis. Immunoprecipitation assays on homogenates of microinjected oocytes showed that labeled 5S RNA can become complexed either with L5 or with TFIIIA. Nucleotides 11 through 108 of the 5S RNA molecule provide the necessary sequence and conformational information required for the formation of immunologically detectable complexes with TFIIIA or L5 and for nuclear accumulation. Furthermore, labeled 5S RNA from microinjected 7S RNPs can subsequently become associated with L5. Such labeled 5S RNA is found in both 5S RNPs and 7S RNPs in the cytoplasm, but only in 5S RNPs in the nucleus of microinjected oocytes. These data suggest that during oogenesis a major pathway for incorporation of 5S RNA into nascent ribosomes involves the migration of 5S RNA from the nucleus to the cytoplasm for storage in an RNP complex with TFIIIA, exchange of that protein association for binding with ribosomal protein L5, and a return to the nucleus for incorporation into ribosomes as they are being assembled in the amplified nucleoli.     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.